Saccharification Property Of Poplar Stems With Modified Lignin By RNAi Technique

Biofuels are gaining increased public and scientific attention, driven by the desires to improve air quality and secure energy supply. Considering high-cost in biodiesel production, ethanol becomes the most promising biofuel. Ethanol from food stocks has implications of world food prices and limited energy yield, thus the technology has led to the development of cellulosic ethanol. To relieve the energy crisis under food security, forest bioenergy is thus inevitably focused on. Lignocellulose is the largest renewable resource on earth. In China, if the pipeline of lignocellulose bioconversion to ethanol and the energy feedstock based on foresty are established, the purpose of "not competing with food" and "not competing with lands" can be achieved with economic, environmental and social benefits. Poplar, as a model tree species, is widely cultivated globally as the important plantation trees, and China is the world's largest in poplar plantation. The poplar genus is widely distributed, together with its asexual reproduction ability, fast growth and high yield, could minimize the competition between biofuels and food crops. In addition, the sequenced genome of ploplar exhibits more attractive for advanced breeding as an alternative biofuel source. However, efficient hydrolysis of lignocellulose is the biggest technical challenge on forest bionergy development because lignin is a major limitation for converting lignocellulose to ethanol in poplar.We are attempting to genetically modify poplar to decrease lignin content in order to enhance fermenTab. sugars which can be converted to ethanol. To do this, we targeted at coumaroyl shikimate 3-hydroxylase ( C3H ) , cinnamate 4-hydroxylase ( C4H ) and hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase(HCT)involved in lignin biosynthesis using RNAi technique. Four full gene-length cDNAs of C3H1, C4H1, HCT1 and HCQ3 in Populus tomentosa Carr. were cloned using in silico and molecular technique. The open reading frame of C3H1, HCT1, HCQ3 and C4H1 gene encodes a peptide of 508, 432, 431 and 505 amino acids respectively. RNAi vectors expressing ds-RNA for each of them were constructed. Additional fifteen multi-gene fusion RNAi constucts with different combinations of the four genes used for transformation were obtained by employing a Gateway-based platform(pBIRNAi-C3H1R-i-C3H1L,pBIRNAi-C4H1R-i-C4H1L,pBIRNAi-HCT1R-i-HCT1L and pBIRNAi-HCQ3R-i-HCQ3L) Poplar was transformed via the Agrobactria-mediated leaf-disc method. 30 independent transgenic poplar lines harboring the three gene RNAi constructs were obtained and vegetatively propagated by cutting for each lines in the greenhouse.The kanamycin-resistant seedlings were subjected to further analyses of gene expression by real-time PCR. The transcription level of C3H1 and HCQ3 in both non- and transgenic lines were analyzed by real-time PCR. 89.04%, 82.22% and 68.38% reduction were obtained in the C3H1 RNAi inhibition lines 323, 325 and 322 respectively in comparison with the non-transgenic controls, and 67.64%, 56.35%, 49.88% and 45.05% reduction were obtained in the HCQ3 RNAi inhibition lines 312, 308, 502 and 307.Stem cross-section staining and microstructure observations showed that cell layers of the secondary xylem in transgenic plants were increased, but the cell became smaller with cell wall collapsed and irregular thickened secondary wall. It indicated that the xylem development and lignin deposition pattern in transgenic plants were changed. Lignin and cellulose content test showed that transgenic plants with reduced lignin content generally in accordance with the transcript level of the target gene. Lignin reduction in transgenic plants also showed higher cellulose content.To determine relationships between lignin content and the efficiency of chemical/enzymatic saccharification, stem material with modified lignin content were tested. Solubilized total sugars in extractive free cell wall residues and hydrolyzates were estimated spectrophotometrically using the phenol-sulfuric acid assay. Monomeric sugars in hydrolysates from acid pre-treatment and the glucose and xylose contents of enzymatic hydrolysates were determined by HPLC. Plants with the least lignin had the highest total carbohydrate levels in untreated biomass, re?ecting compensation for the reduction in lignin level on a mass balance basis. After 72 h incubation, saccharification efficiency was higher in C3H and HCT reduced lines compared with controls. More than 90% of the released sugar from most lines was glucose, indicating enzymatic hydrolysis of cellulose. Enzymatic hydrolysis released more xylose from transgenic lines than from control lines, suggesting that lignin modification increases the accessibility of residual hemicellulose to degradative enzymes.This study tested the effectiveness of reduction of lignin content by RNAi strategy using several key genes and their fusion constructs. The results indicated that lignin is probably the major factor in recalcitrance of cell walls to saccharification. Moreover, it demonstrated that genetic reduction of lignin content effectively overcame cell wall recalcitrance to bioconversion. This approach could obviate the need for acid pretreatment, as indicated by that the saccharification efficiency of untreated biomass of the 312 and 323 line were even greater than that of control plants with pretreatment. The genes targeted in the present work thus would be the candidates for improving saccharification in other bioenergy plants.